Widely conserved AHL transcription factors are essential for NCR gene expression and nodule development in Medicago

Symbiotic nitrogen fixation by Rhizobium bacteria in the cells of legume root nodules alleviates the need for nitrogen fertilizers. Nitrogen fixation requires the endosymbionts to differentiate into bacteroids which can be reversible or terminal. The latter is controlled by the plant, it is more beneficial and has evolved in multiple clades of the Leguminosae family. The plant effectors of terminal differentiation in inverted repeat-lacking clade legumes (IRLC) are nodule-specific cysteine-rich (NCR) peptides, which are absent in legumes such as soybean where there is no terminal differentiation of rhizobia. It was assumed that NCRs co-evolved with specific transcription factors, but our work demonstrates that expression of NCR genes does not require NCR-specific transcription factors. Introduction of the Medicago truncatula NCR169 gene under its own promoter into soybean roots resulted in its nodule-specific expression, leading to bacteroid changes associated with terminal differentiation. We identified two AT-Hook Motif Nuclear Localized (AHL) transcription factors from both M. truncatula and soybean nodules that bound to AT-rich sequences in the NCR169 promoter inducing its expression. Whereas mutation of NCR169 arrested bacteroid development at a late stage, the absence of MtAHL1 or MtAHL2 completely blocked bacteroid differentiation indicating that they also regulate other NCR genes required for the development of nitrogen-fixing nodules. Regulation of NCRs by orthologous transcription factors in non-IRLC legumes opens up the possibility of increasing the efficiency of nitrogen fixation in legumes lacking NCRs.